Part:BBa_K5375003

pPICZαA-HSP70-sfGFP

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 383
  Illegal BglII site found at 1307
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 468
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 358
  Illegal SapI.rc site found at 1677
  Illegal SapI.rc site found at 1966
  

Origin

Synthesized by company and constructed by the team.

Properties

Fusion expression of protein HSP70-GFP.

Usage and Biology

The pPICZαA vectors are 3.6 kb plasmids designed for the expression and secretion of recombinant proteins in *Pichia pastoris* yeast. These recombinant proteins are expressed as N-terminal fusions with the coding sequence of the α-factor secretion signal derived from *Saccharomyces cerevisiae*. The vectors facilitate high-level gene expression induced by methanol in *Pichia pastoris* and can be utilized with any strain of this yeast. We inserted the HSP70 coding gene into this plasmid for fusion expression with GFP.

Cultivation, Purification and SDS-PAGE

Figure 1. Plasmid diagram of pPICZαA-HSP70-sfGFP.

The vector pPICZαA originates from the *Pichia pastoris* expression vector. It is methanol-inducible and allows high-level expression and secretion of recombinant proteins, specifically in yeast. This vector was chosen to measure the DNA expression of HSP70 in yeast.

Figure 2. PCR amplification of the fragment used for plasmid construction.

We constructed pPICZαA-HSP70-sfGFP using homologous recombination. The HSP70-sfGFP sequence was amplified by PCR with a length of 2123 bp.

Figure 3. Growth of plasmid pPICZaαA-HSP70 transformed bacteria on LB agar plates.

Secondly, the correct plasmids of pPICZαA-HSP70-sfGFP were transferred into yeast. They were cultured on YPD medium agar plates overnight. The image shows significant bacterial growth on YPD agar plates with easily observable individual colonies.

Figure 4. Growth of pPICZαA-HSP70-sfGFP in yeast on YPD agar plates.

Finally, the protein expression of pPICZαA-HSP70 was tested by SDS-PAGE. The results are shown below, displaying the target band for HSP70 (78 kDa).

Figure 5. SDS-PAGE analysis of HSP70 expression in yeast cells.

Measurement and Characterization

We used DNA sequencing to determine the full nucleotide sequence of our reconstructed plasmids, verifying the accuracy and integrity of the pPICZαA-HSP70-sfGFP plasmids.

Figure 6. Sanger sequencing result of pPICZαA-HSP70-sfGFP plasmid.

Reference

[1](https://www.thermofisher.cn/order/catalog/product/cn/zh/V19520)

[2](https://www.snapgene.com/plasmids/yeast_plasmids/pPICZ(alpha)_)